JP2022552488A - Glutathione-producing yeast strain and method for producing glutathione using the same - Google Patents

Abstract

A novel glutathione-producing yeast strain and a method for producing glutathione using the same are provided. Kind Code: A1 The present application relates to a novel glutathione-producing yeast strain and a method for producing glutathione using the same.

Description

The present application relates to a novel glutathione-producing yeast strain and a method for producing glutathione using the same.

Glutathione (GSH) is the most commonly occurring organic sulfur compound in cells and is a tripeptide consisting of three amino acids: glycine, glutamate, and cysteine. form.

Glutathione exists in the body in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Reduced glutathione (GSH), which exists in a relatively high proportion under general conditions, is mainly distributed in the liver and skin cells of the human body, and has an antioxidant function that decomposes and removes active oxygen. It plays an important role such as detoxification such as removing exogenous compounds and whitening by suppressing the production of melanin pigment.

As aging progresses, the production of glutathione gradually decreases, and the reduction in the production of glutathione, which plays an important role in antioxidant and detoxification, promotes the accumulation of active oxygen, which is the main cause of aging. (Non-Patent Document 1).

Glutathione, which has such various functions, has been spotlighted as a material in various fields such as pharmaceuticals, functional foods, and cosmetics, and is also used in the production of taste-imparting materials, food and feed additives. Glutathione is known to have a significant effect of improving the taste of the original material and maintaining the taste persistence, and is used as a kokumi flavor enhancer when used alone or mixed with other substances. there is Kokumi materials are usually richer than conventional umami materials such as nucleic acids and MSG, and are known to be produced by decomposition and aging of proteins.

However, despite the increasing demand for glutathione, which can be used in various fields, the industrial production of glutathione is very costly, and the market is not sufficiently activated. is.

Sipes IG et al, The role of glutathione in the toxicity of xenobiotic compounds: metabolic activation of 1,2-dibromoethane by glutathione, Adv Exp Med Biol. 1986;197:457-67. Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 Rice et al., 2000, Trends Genet. 16: 276-277 Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994 [CARILLO ETA/.](1988) SIAM J Applied Math 48: 1073 Smith and Waterman, Adv. Appl. Math (1981) 2:482 Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) Gribskov et al (1986) Nucl. Acids Res. 14: 6745 http://www.ncbi.nlm.nih.gob/blast/

The present inventors have made diligent efforts to solve the above problems, and as a result, have developed a novel strain with excellent glutathione-producing ability and completed the present application.

The present application aims to provide novel strains of Saccharomyces cerevisiae that produce glutathione.

Another object of the present application is to provide a method for producing glutathione, which includes the step of culturing the strain.

Furthermore, the present application relates to the step of culturing the strain, and mixing at least one substance selected from the cultured strain, its dried product, extract, culture, crushed product, and glutathione recovered therefrom and an additive. The object of the present invention is to provide a method for producing a glutathione-containing composition, comprising the steps of:

Furthermore, the present application relates to strains, dried products, extracts, cultures and lysates of said strains, and glutathione recovered from at least one of said strains, dried products, extracts, cultures and lysates. An object of the present invention is to provide a composition for antioxidant function, detoxification, immunity enhancement, cosmetic, food and feed containing at least one substance selected from the group consisting of.

Furthermore, the present application relates to strains, dried products, extracts, cultures and lysates of said strains, and glutathione recovered from at least one of said strains, dried products, extracts, cultures and lysates. An object of the present invention is to provide a pharmaceutical composition or a pharmaceutical composition for use in the prevention or treatment of diseases caused by glutathione deficiency, containing at least one substance selected from the group consisting of:

The strains of the present application are capable of producing significantly higher amounts of glutathione than conventional glutathione-producing strains. Therefore, the strains of the present application are useful for producing glutathione.

In addition, the group consisting of the strain of the present application, the dried product, extract, culture, crushed product of the strain, and glutathione recovered from at least one of the strain, dried product, extract, culture, crushed product A composition containing at least one substance selected from has an excellent ability to remove active oxygen, and is therefore useful as a composition for antioxidant function, detoxification, and immunity enhancement.

Thus, said composition is also useful for cosmetic compositions, pharmaceutical compositions and their manufacture.

In addition, when the composition is used in food, it not only has the functions described above, but also greatly improves the taste of the food.

Saccharomyces cerevisiae CEN. FIG. 2 shows sensory evaluation results of food compositions containing extracts of PK2-1D, CJ-5 and CJ-37 strains. Saccharomyces cerevisiae CEN. FIG. 2 shows the results of confirming the radical scavenging ability of extracts of PK2-1D, CJ-5 and CJ-37 strains. Saccharomyces cerevisiae CEN. FIG. 2 shows the results of confirming the active oxygen absorption capacity of PK2-1D, CJ-5, and CJ-37 strains.

These will be specifically described below. It should be noted that each description and embodiment disclosed in this application also applies to other descriptions and embodiments, respectively. That is, all combinations of the various elements disclosed in this application are included in this application. Also, the present application is not limited to the following specific descriptions.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. be. Moreover, equivalents thereof are also intended to be included in this application.

One aspect of the present application provides novel Saccharomyces cerevisiae strains that produce glutathione.

The glutathione-producing strain of the present application may be the Saccharomyces cerevisiae CJ-5 strain with accession number KCCM12568P.

The glutathione-producing strain of the present application is 90% or more the 18S (ITS, 5.8S) rRNA sequence (SEQ ID NO: 3) of other Saccharomyces cerevisiae of the same genus and species, specifically Saccharomyces cerevisiae or Saccharomyces cerevisiae YJM1592, Specifically, those containing 18S (ITS, 5.8S) rRNA sequences with 91%, 92%, 93%, 93.2%, 93.5% or 93.7% or more homology or identity Although there may be, it is not limited to these. Furthermore, the glutathione-producing strains of the present application have a 18S (ITS, 5.8S) rRNA sequence of YJM1592 of 90% or more, specifically 91%, 92%, 93%, 93.2%, 93.5% or 18S (ITS, 5.8S) rRNA sequences with 93.7% or more homology or identity and less than 96%, 95.5% or 95.2% identity Good, but not limited to these. The 18S (ITS, 5.8S) rRNA sequence is an ITS (Internally transcribed spacer) sequence present between 18S rRNA and 5.8S, and is a sequence used for species classification.

A glutathione-producing strain of the present application may have the 18S (ITS, 5.8S) rRNA sequence of SEQ ID NO:1. According to one embodiment, the 18S (ITS, 5.8S) rRNA sequence of said strain is more than 90%, specifically 95%, 96%, SEQ ID NO:1. Those having 97%, 98% or 99% or more homology or identity, but are not limited to these.

"Homology" or "identity" as used in this application means the degree to which two given amino acid or base sequences are related, and is expressed as a percentage. Homology and identity are often used interchangeably.

Conserved polynucleotide or polypeptide sequence homology or identity may be determined by standard sequence algorithms, together with default gap penalties established by the program used. Substantially homologous or identical sequences generally have at least about 50%, 60%, 70%, 80% or 90% or more hybridize. The hybridization may be such that the polynucleotide has degenerate codons in place of codons.

Whether two arbitrary polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using default parameters such as those described in Non-Patent Document 2 and known computer algorithms such as the "FASTA" program. can decide. Alternatively, Needleman-Unsch (Needleman- Wunsch) algorithm (Non-Patent Document 4) (including GCG program package (Non-Patent Document 5), BLASTP, BLASTN, FASTA (Non-Patent Documents 6, 7, 8)). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

Polynucleotide or polypeptide homology, similarity or identity is determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 9. can do. Briefly, the GAP program defines it as the number of similar sequence symbols (ie, nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program are: (1) a binary comparison matrix (identity takes a value of 1, non-identity takes a value of 0) and the weights of (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or gap open penalty of 10 , gap extension penalty 0.5) and (3) no penalty for end gaps.

Further, whether or not any two polynucleotide or polypeptide sequences have homology, similarity or identity is confirmed by comparing the sequences in Southern hybridization experiments under defined stringent conditions. The defined suitable hybridization conditions are within the skill in the art and are determined by methods well known to those of ordinary skill in the art.

The term "glutathione" in the present application is used interchangeably with "glutathione" and "GSH", and is composed of three amino acids: glutamate, cysteine and glycine. means peptide. Glutathione is used as, but not limited to, raw materials for pharmaceuticals, functional foods, flavoring materials, foods, feed additives, cosmetics, and the like.

The novel Saccharomyces cerevisiae strains of the present application are characterized by a high glutathione-producing capacity.

The strain may be a strain with enhanced glutathione-producing ability, a strain with high glutathione-producing ability, or a strain with improved glutathione-producing ability.

"Glutathione-producing strain" in the present application is used interchangeably with terms such as "strain having glutathione-producing ability" and "glutathione-producing strain".

The Saccharomyces cerevisiae strain according to the present invention contains 0.6% by weight or more, specifically 0.7% by weight or more, 0.8% by weight or more, 0.9% by weight or more, based on the dry cell weight. It may contain 0% by weight or more and 1.1% by weight or more of glutathione, but is not limited to these.

Another aspect of the present application provides a composition for producing glutathione comprising said strain.

"Glutathione production" or "glutathione production" in the present application includes accumulating glutathione in cells.

The glutathione-producing composition is a composition that produces glutathione using the strain of the present application. For example, the composition contains the strain and uses the strain to produce glutathione. good.

The glutathione-producing composition of the present application may further comprise any suitable additive commonly used in the glutathione-producing composition. The additive may be naturally occurring or non-naturally occurring, but is not limited thereto.

Said additives include excipients and/or emulsifiers. The above-mentioned excipients can be appropriately selected and used according to the intended use and form. Aluminum Silicate, Calcium Monohydrogen Phosphate, Calcium Sulfate, Sodium Chloride, Sodium Bicarbonate, Refined Lanolin, Dextrin, Sodium Alginate, Methylcellulose, Colloidal Silicon Dioxide, Hydroxypropyl Starch, Hydroxypropyl Methylcellulose, Propylene Glycol, Casein, Calcium Lactate. , primogel, gum arabic, specifically at least one component selected from starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol, maltodextrin, but limited to these not to be Emulsifiers include glycerin esters, sorbitan esters, monoglycerides, diglycerides, triglycerides, sucrose esters, sorbitan esters, propylene glycol esters, glycerin fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, sucrose fatty acid esters, lecithin, mixtures thereof, and the like. are used, but are not limited to these, and those known in the art can be used as appropriate.

Examples of the excipient include, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents, tonicity agents and the like.

Yet another aspect of the present application provides a method for producing glutathione comprising culturing the strain. Glutathione may be accumulated in the cells by culturing the strain.

The medium and other culture conditions used for culturing the strain of the present application may be any medium that is commonly used for culturing Saccharomyces microorganisms, specifically suitable carbon sources, nitrogen sources, and phosphorus sources. , inorganic compounds, amino acids and/or vitamins, under aerobic or anaerobic conditions, the strain of the present application can be cultured by adjusting the temperature, pH and the like.

Examples of the carbon source in the present application include carbohydrates such as glucose, fructose, sucrose and maltose, sugar alcohols such as mannitol and sorbitol, organic acids such as pyruvic acid, lactic acid and citric acid, amino acids such as glutamic acid, methionine and lysine. Examples include, but are not limited to. Alternatively, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, corn steep liquor can be used, as well as glucose and pasteurized pretreated molasses (i.e., converted to reducing sugars). Carbohydrates such as molasses) can be used, as can any other suitable carbon source. These carbon sources can be used alone or in combination of two or more.

The nitrogen sources include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, amino acids, peptone, NZ-amine, meat extracts, yeast extracts, and malt extracts. , corn steep liquor, casein hydrolysate, fish or its degradation products, defatted soybean cake or its degradation products, and organic nitrogen sources can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

Examples of the phosphorus source include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium-containing salts corresponding thereto. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate.

Besides that, the medium can contain amino acids, vitamins and/or suitable precursors and the like. Specifically, an L-amino acid or the like can be added to the culture medium for the strain. Specifically, glycine, glutamate and/or cysteine can be added, and if necessary, L-amino acids such as lysine can be further added. , but not necessarily limited to these.

The medium or precursors can be added to the culture in batches or continuously, but are not limited thereto.

The pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture in a suitable manner during cultivation of the strains in the present application. In addition, during the culture, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress the generation of air bubbles. Furthermore, oxygen or an oxygen-containing gas may be injected into the culture to maintain the aerobic state of the culture, or no gas may be injected to maintain the anaerobic and microaerobic conditions. , nitrogen, hydrogen or carbon dioxide gas may be injected.

The temperature of the culture may be 25°C to 40°C, more specifically 28°C to 37°C, but is not limited thereto. The culture period is continued until the desired production amount of the useful substance is obtained, and is specifically from 1 hour to 100 hours, but is not limited thereto.

The method for producing glutathione may further include an additional step after the culturing step. The additional step is appropriately selected depending on the use of glutathione.

Specifically, the method for producing glutathione may include, after the culturing step, recovering glutathione from at least one substance selected from the strain, its dried product, extract, culture, and crushed product. .

"Culture" in this application means a product obtained by culturing the strain of this application. For example, the strain is cultured in a medium to take in nutrients, and includes a medium containing by-products generated by substance metabolism. Dilutions and concentrates of the medium, dried products obtained by drying the medium, It is a culture of any form that can be formed using the medium, such as crudely purified products, purified products, and mixtures thereof. The culture of the microorganism of the present application may or may not contain the microorganism. Culture is as described above.

The “crushed product” in the present application may be a product obtained by crushing or lysing the strain or its culture, or a supernatant obtained by centrifuging the crushed product.

Said cultures, lysates, their supernatants and their fractions are also included in the present application. The method may further comprise lysing the strain prior to or concurrently with the recovering step. Lysis of strains can be performed using methods commonly used in the technical field to which the present application belongs, such as lysis buffer, sonicator, heat treatment, French press, and the like. The lysis step includes, but is not limited to, enzymatic reactions such as cell wall-degrading enzymes, nucleases, nucleotransferases, and proteinases.

For the purposes of this application, dry yeast containing a high content of glutathione by the method for producing glutathione, yeast extract, yeast extract mix powder, purely purified glutathione ( pure glutathione) is produced, but is not limited to these, and is produced as appropriate according to the intended product.

Dry yeast in this application is used interchangeably with terms such as "dry strain". The dry yeast can be produced by drying yeast cells that have accumulated glutathione, and is specifically included in feed compositions, food compositions, etc., but is limited thereto. is not.

Yeast extract in this application is used interchangeably with terms such as "strain extract". The strain extract means a substance remaining after separating the cell wall from the cells of the strain. Specifically, it means the remaining component after removing the cell wall from the component obtained by lysing the cells. The strain extract contains glutathione, and components other than glutathione include, but are not limited to, at least one of proteins, carbohydrates, nucleic acids, and fibers.

In the recovery step, the target substance, glutathione, can be recovered by a suitable method known in the art.

The recovering step may include a purification process. The purification step may be a step of separating and purifying only glutathione from the strain. Through the purification process, pure glutathione can be produced.

Optionally, in the method for producing glutathione, after the culturing step, a substance selected from the obtained strain, its dried product, extract, culture, crushed product, and glutathione recovered therefrom are mixed with additives. may further include the step of: Through the mixing step, a yeast extract mix powder can be produced.

Said additives may include excipients and/or emulsifiers. Excipients and emulsifiers can be appropriately selected and used by those skilled in the art, and examples thereof are as described above.

Still another aspect of the present application is the step of culturing the strain, and mixing a substance and an additive selected from the cultured strain, its dried product, extract, culture, crushed product, and glutathione recovered therefrom. A method for producing a composition comprising glutathione is provided, comprising the steps of:

Compositions of the present application may further comprise naturally occurring or non-naturally occurring materials. The substances include, but are not limited to, pharmaceutically acceptable carriers, food acceptable carriers, cosmetically acceptable carriers, etc., depending on the intended use of the composition. not something. The carrier can be appropriately selected by those skilled in the art based on known contents.

In the composition, the content of the substance selected from the strain of the present application, its dried product, extract, culture, crushed product, and glutathione recovered therefrom relative to the total weight of the composition It may be 3 to 30% by weight.

Said additives may include excipients and/or emulsifiers. Excipients and emulsifiers can be appropriately selected and used by those skilled in the art, and examples thereof are as described above.

Still another aspect of the present application is recovered from the strain, the dried product, extract, culture, crushed product of the strain, and at least one of the strain, dried product, extract, culture, crushed product A composition for antioxidant function, detoxification, immunity enhancement, cosmetic, food or feed, comprising at least one substance selected from the group consisting of glutathione and glutathione.

Still another aspect of the present application is recovered from the strain, the dried product, extract, culture, crushed product of the strain, and at least one of the strain, dried product, extract, culture, crushed product Provided is a method for producing a composition for antioxidant function, detoxification, immunity enhancement, cosmetic, food or feed, comprising at least one substance selected from the group consisting of glutathione and glutathione.

For the purposes of this application, said composition may comprise the strain itself, including culture of said strain, dried product of said strain, extract of said strain, homogenate of said strain. Alternatively, it may contain glutathione recovered from the culture, extract, dried product, or crushed product of the strain, but is not limited to these. That is, since the strain, its dried product, extract, culture, and crushed product contain glutathione, depending on the specific use of the composition, even if the form containing glutathione is slightly different, the desired glutathione can be obtained. Any form may be used as long as it increases the content.

In the composition, the content of the substance selected from the strain of the present application, its dried product, extract, culture, crushed product, and glutathione recovered therefrom relative to the total weight of the composition It may be 3 to 30% by weight. The composition may also further comprise any suitable additive normally used in compositions.

The composition comprises a glutathione-producing strain of the present application, a dried product, extract, culture, crushed product of the strain, and at least one of the strain, dried product, extract, culture, crushed product. The glutathione content may be increased by containing at least one substance selected from the group consisting of recovered glutathione.

Still another aspect of the present application is recovered from the strain, the dried product, extract, culture, crushed product of the strain, and at least one of the strain, dried product, extract, culture, crushed product and glutathione.

The term "antioxidant" as used in the present application broadly means the action of suppressing, reducing or controlling the production of oxidation reactions that occur in nature, and more specifically, free radicals produced in the body, from the free radicals It means the action of suppressing, reducing or controlling the generation or reaction of hydrogen peroxide or peroxides produced, or the hydroxyl radicals produced from the hydrogen peroxide.

Still another aspect of the present application is recovered from the strain, the dried product, extract, culture, crushed product of the strain, and at least one of the strain, dried product, extract, culture, crushed product and glutathione.

"Detoxification" in this application includes all actions that eliminate or reduce toxicity due to harmful substances.

Glutathione is an endogenous antioxidant produced by cells that is directly involved in neutralizing free radicals and reactive oxygen compounds, as well as maintaining exogenous antioxidants such as vitamins C and E in their reduced form. Therefore, the detoxification includes the action of suppressing, reducing, or controlling the generation or reaction of active oxygen or free radicals, hydrogen peroxide or peroxides generated from the free radicals, and hydroxyl radicals generated from the hydrogen peroxide. is included. Glutathione can also protect the thiol groups of cellular proteins, prevent side effects from drug overdoses such as acetaminophen, and detoxify methylglyoxal, which is produced as a metabolic byproduct. As used, said detoxification also includes mitigating or reducing the toxicity of the compound.

Still another aspect of the present application is recovered from the strain, the dried product, extract, culture, crushed product of the strain, and at least one of the strain, dried product, extract, culture, crushed product and glutathione.

"Immune enhancement" in the present application means improving the ability of the body to defend itself against antigens, specifically means increasing cellular and humoral immunity against antigens, and more specifically, It means that the immune function is improved compared to before administration of the composition. Mechanisms for enhancing immunity include, but are not limited to, promoting the activity of antigen-presenting cells such as macrophages, or promoting specific activity against lymphocytes.

As described above, glutathione has strong antioxidant power, improves liver function, plays a role in helping to decompose toxic substances in the body, and participates in the immune system by removing harmful active oxygen. That is, glutathione increases the activity of T lymphocytes and leukocytes, thereby improving the immunity of the body and restoring the physical strength of individuals weakened by various chronic diseases. At least one substance selected from the group consisting of dried product, extract, culture, crushed product of the strain, and glutathione recovered from at least one of the strain, dried product, extract, culture, crushed product, , is useful for enhancing immunity.

Still another aspect of the present application is recovered from the strain, the dried product, extract, culture, crushed product of the strain, and at least one of the strain, dried product, extract, culture, crushed product and glutathione.

"Cosmetic compositions" in this application include solutions, external ointments, creams, foams, nutritional lotions, softening lotions, packs, softening waters, milky lotions, makeup bases, essences, liquid cleansers, bath additives, sunscreens. selected from the group consisting of creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays However, it is not limited to these dosage forms.

The cosmetic composition may further contain one or more cosmetically acceptable carriers blended in general skin cosmetics, and typical ingredients include, for example, oil, water, surfactants, moisturizers, Lower alcohols, thickeners, chelating agents, dyes, preservatives, fragrances, and the like may be added as appropriate, but are not limited to these. A cosmetically acceptable carrier contained in the cosmetic composition of the present application can be appropriately selected by those skilled in the art according to the dosage form of the cosmetic composition.

Still another aspect of the present application is recovered from the strain, the dried product, extract, culture, crushed product of the strain, and at least one of the strain, dried product, extract, culture, crushed product and glutathione.

As an example, the food composition may comprise cells or an extract of the strain of the present application, more specifically, added to the strain extract depending on the food product to which it is applied. It is intended to be used after being formulated into an appropriate form by containing an agent, but is not limited to these. The strain extract is as described above.

Said additives may include excipients and/or emulsifiers. Excipients and emulsifiers can be appropriately selected and used by those skilled in the art, and examples thereof are as described above. Also included are food supplement additives, food stabilizers, moisture preservatives, and the like.

The "food composition" in this application includes all forms of functional food, nutritional supplements, health food, food additives, and the like.

Food compositions of the above type can be manufactured in various forms by conventional methods known in the art.

The food compositions include forms such as pills, powders, granules, infusions, tablets, capsules, and liquids. Foods to which the composition of the present application is added include, for example, rice, edible grain flour ), Grain soup, Rice bowl, Noodles, Bowser, Instant rice, Seasoning, Bento rice, Dried rice, Bread, Edible sugar, Rice cake, Sauce, Sauce, Spices, Salt, Condiment, Condiment powder, Processing, Freezing, Drying and cooked fruits and vegetables, jellies, jams, candied fruits, eggs, milk and other dairy products, edible fats and oils, coffee, cocoa, coffee substitutes, tapioca, flour and grain preparations, noodles, udon, noodles, kalguksu, Various foods such as cold noodles, porridge, soup, soup, instant food (instant food), frozen food, retort food, other beverages, gum, tea, vitamin complexes, and health supplements. It is not limited.

The food compositions of the present application may contain various herbal extracts, food supplements, natural carbohydrates, etc. as additional ingredients, similar to ordinary food. The food supplementary additives may also include food supplementary additives commonly used in the art, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like.

Examples of the natural carbohydrates include ordinary sugars including monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. mentioned. As flavoring agents other than those mentioned above, it is advantageous to use natural flavoring agents (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). Ordinary food additives such as nucleic acids, amino acids, organic acids, etc., which are used for the purpose of replenishing taste and nutrition, may also be added.

In addition to the above, the food composition of the present application contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, coloring agents and fillers (cheese, chocolate, etc.), pectin. Acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloids, thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, etc. may be contained. . In addition, it may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used alone or in combination.

The food of the present application can be produced by a method commonly used in the technical field, and can be produced by adding raw materials and components that are commonly added in the technical field. Moreover, the dosage form of the food is not limited as long as it is a dosage form recognized as a food.

In addition, when the food composition of the present application is used as a functional food, the food composition of the present application can be manufactured into various dosage forms, and unlike general drugs, food is used as a raw material, The food of the present application can be ingested as a supplement because it has the advantage of not causing side effects that may occur when taking medicine for a long period of time and is excellent in portability.

On the other hand, the food compositions of the present application may contain flavoring agents, flavoring agents, coloring agents, fillers, stabilizing agents and seasoning materials, which are also classified as food additives.

A "flavor" in the present application is a material added to improve the flavor of food. The seasoning material may be a material that imparts excellent taste to foods.

The seasoning ingredients are classified according to taste components. That is, they are classified into neutral seasoning materials, beef flavor seasoning materials, chicken seasoning materials, pork seasoning materials, kokumi seasoning materials, etc. according to flavor.

'Kokumi flavor' means a seasoning material that produces a rich taste, and the above 'kokumi' is a word derived from Japanese, and in the English-speaking world it means 'mouthfulness', 'continuity', and 'continuity'. It is also expressed as ``thickness'' and ``heartiness'', and in Korean it is expressed as ``rich taste'', ``thick taste'', ``taste that spreads in the mouth'', ``rich taste'', and ``taste with a strong taste''. means taste. "Neutral flavor" means a seasoning material that maximizes umami and minimizes other flavors to produce a mellow and refreshing flavor. Oils and oils such as grapeseed oil are said to have a neutral flavor. “Tasting” means having sourness, sweetness, saltiness, bitterness, umami, etc., but is not limited to these.

The food composition of the present application may contain 3 to 30% by weight of the yeast extract of the present application relative to the total weight of the composition, and is selected from ammonium chloride, maltodextrin, powdered glucose and an emulsifier. It may further contain the above components. In one embodiment, the food composition comprises 3-25% by weight of yeast extract, 20-35% by weight of ammonium chloride, 25-35% by weight of maltodextrin, 3% by weight of powdered glucose, relative to the total weight of the composition. ~7% by weight, and may include, but is not limited to, 0.1-1% by weight of emulsifier.

Still another aspect of the present application is recovered from the strain, the dried product, extract, culture, crushed product of the strain, and at least one of the strain, dried product, extract, culture, crushed product and glutathione.

Specifically, the feed composition contains at least one of the strain itself, the dried strain product, and the strain extract of the present application, or contains a cell wall component obtained in the process of producing the extract of the strain. including but not limited to dry yeast and/or yeast extract.

A "feed composition" of the present application is any natural or artificial diet, meal, etc., or component of said meal for or suitable for eating, ingestion, and digestion by an animal; The feed containing yeast with increased glutathione content according to the application can be manufactured into various forms of feed known in the art, specifically into concentrate feed, rough feed and/or special feed. can.

The type of feed is not particularly limited, and feeds commonly used in the art can be used. Examples of the feed include vegetable feeds such as cereals, roots and fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, meals, cereal by-products, and proteins. , inorganic substances, minerals, fats and oils, single-cell proteins, zooplankton, and animal feeds such as food and drink, but are not limited to these. These can be used alone or in combination of two or more.

Yet another aspect of the present application includes glutathione recovered from at least one of the strain, its dried product, extract, culture, lysate, said strain, dried product, extract, culture, lysate, Provided is a composition for manufacturing pharmaceuticals that is used for the prevention or treatment of diseases caused by glutathione deficiency.

Yet another aspect of the present application includes glutathione recovered from at least one of the strain, its dried product, extract, culture, lysate, said strain, dried product, extract, culture, lysate, Provided is a pharmaceutical composition for use in preventing or treating diseases caused by glutathione deficiency.

A "pharmaceutical composition" in this application means a chemical or biological compound or substance, or two or more of such compounds or substances, for use in the medical diagnosis, therapy, treatment or prevention of a disease or pathology. mixture or combination.

A "composition for pharmaceutical manufacture" in this application is used interchangeably with a pharmaceutical composition or means a composition further comprising any additional ingredients necessary for pharmaceutical manufacture and/or formulation. However, it is not limited to these.

As described above, glutathione contributes to the antioxidant function of decomposing and removing active oxygen, detoxification, and enhancement of immunity. Contained in concentrations, it is useful in the manufacture of medicaments, said medicaments being useful for treating diseases due to glutathione deficiency.

In addition, since the strains, their cultures, dried products, extracts, and crushed products contain glutathione at high concentrations, they themselves are included in the components of pharmaceutical compositions for the prevention or treatment of diseases caused by glutathione deficiency. may

The disease is not limited as long as it is caused by glutathione deficiency. For example, any disease caused by the accumulation of active oxygen and/or weakened immune system may be used. Specifically, arteriosclerosis, Lou Gehrig's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and Degenerative neurological diseases, including Huntington's disease, myocardial infarction, angina pectoris, coronary artery disease, cardiovascular diseases, including ischemic heart disease, ischemic brain diseases, including stroke, digestive diseases, including diabetes, gastritis and gastric cancer System diseases, cancer, leukemia, cataract, aging, rheumatoid arthritis, hepatitis, atopic dermatitis, etc., but not limited to these.

Said medicament and/or pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" in this application means the property of not being toxic to cells or humans exposed to the pharmaceutical agent. The carrier may be any known in the art, such as buffering agents, preservatives, soothing agents, solubilizers, tonicity agents, stabilizers, bases, excipients, lubricants, and the like. Anything is fine.

In addition, the pharmaceuticals and/or pharmaceutical compositions can be prepared in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, and suppositories by conventional methods. It is used in the form of pharmaceutical agents and sterile injectable solutions. Furthermore, they are used in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or gel-like skin preparations for external use. Carriers, excipients and diluents contained in the pharmaceuticals of the present application include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate. , cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. For formulation, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants and surfactants are used.

Oral solid dosage forms include tablets, pills, powders, granules, capsules, etc., wherein these solid dosage forms contain at least one excipient in the composition, such as starch, calcium carbonate, and the like. , sucrose, lactose, gelatin, etc. are mixed. Besides ordinary excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, Sweeteners, flavoring agents, preservatives and the like may be used. Parenteral formulations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizates and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like.

The medicament and/or pharmaceutical composition is administered in a pharmaceutically effective amount. "Administration" as used in this application means introducing a given substance into an individual in an appropriate manner, and the route of administration of said composition is any common route as long as it can deliver to the target tissue. may be administered. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration include, but are not limited to.

The "individual" means all animals including humans, rats, mice, domestic animals, and the like. Specifically, it may be mammals including humans.

The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not causing side effects, and an effective dose level. is the patient's sex, age, body weight, health condition, type of disease, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, combination or concomitant drugs The factors involved, as well as others known in the medical arts, can be readily determined by those of ordinary skill in the art. For administration, the above recommended dosage may be administered once a day, or may be administered in several doses.

Yet another aspect of the present application provides the use of said strain for glutathione production.

The strain is as described above.

The present application will be described in more detail below with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are merely illustrative of the present application, and the present application is not limited to these Examples and Experimental Examples.

Selection of strains with excellent glutathione-producing ability and confirmation of glutathione-producing ability Strains were obtained from koji containing various strains and improved to select strains with high glutathione-producing ability.

Specifically, grain samples such as rice, barley, mung bean and oat were collected from a total of 20 regions such as Yongin, Icheon, Pyeongtaek and Hwaseong regions in Gyeonggi-do, Korea to prepare koji. The collected cereal samples were pulverized into a paste, wrapped in a cloth and pressed strongly to form a shape, then wrapped in straw and fermented for 10 days, then gradually dried to produce koji. In order to isolate various strains from the produced koji, the following experiments were carried out. 45 ml of saline solution was added to 5 g of koji and pulverized with a mixer. Pure isolation of the yeast strain was subjected to serial dilution, spread on YPD Agar (yeast extract 10 g/L, bacto peptone 20 g/L, glucose 20 g/L, in 1 liter of distilled water) and cultured at 30° C. for 48 hours. Yeast colonies were also streaked into YPD agar by colony morphology and microscopic examination. 25 ml of YPD broth was dispensed into a 250 ml Erlenmeyer flask, inoculated with the purely isolated strain, cultured with shaking (30° C., 200 rpm) for 48 hours, and strain screening was performed by confirming the glutathione production amount.

Random mutations were induced in the isolated strains for improvement of the primary isolated strains. Among the yeast isolated from the koji, a strain with a glutathione production of 15 mg/L was isolated and named CJ-37 strain. The CJ-37 strain was cultured on a solid medium, then inoculated into broth to obtain a culture solution, and the cells were irradiated with UV using a UV lamp. Then, the UV-irradiated culture solution was smeared on a plate medium, and only mutant strains that formed colonies were isolated and recovered, and their glutathione production amounts were confirmed.

As a result, it was confirmed that the maximum glutathione production of the mutant strain was 105 mg / L, and the strain showing the highest glutathione production (105 mg / L) was selected as the glutathione-producing strain and named CJ-5 strain. . It was deposited with the Korean Culture Center of Microorganisms (KCCM), an international depository under the Budapest Treaty, on July 31, 2019 under deposit number KCCM12568P.

Comparison of glutathione-producing ability In order to confirm the glutathione-producing ability of the CJ-5 strain selected in Example 1, three types of conventionally known microorganisms (Table 1) were cultured by the following method, and the GSH-producing ability was determined. were comparatively analyzed. As a control group, the CJ-37 strain obtained in Example 1 was also cultured and comparatively analyzed.

First, each strain was cultured at 30° C. for 1 day and then placed in a 250 ml corner baffled flask containing 25 ml of YPD medium (yeast extract 10 g/L, Bacto peptone 20 g/L, glucose 20 g/L, in 1 liter of distilled water). was inoculated with the strain, 16 hours after the start of culture, amino acids (L-Cysteine, L-Glutamic acid, L-Glycine) were added at a concentration of 20 mM, and the cells were cultured at 30°C and 200 rpm. To measure the concentration of GSH, 500 μl of the culture solution sample containing the cells was lysed at 80° C. and 800 rpm for 10 minutes, and then the concentration of GSH was measured by Abcam GSH Ratio Detection Assay. The OD value was converted to calculate the dry weight, which was used to calculate the GSH content (%) = GSH weight/yeast dry weight. Table 2 shows the results.

As a result of evaluating the GSH-producing ability of five kinds of yeast, it was confirmed that the CJ-5 strain had the highest GSH content per cell. Specifically, the CJ-5 strain is a strain of conventional GSH-producing strains Candida utilis KCCM50667, Kluyveromyces lactis ATCC8585 and Saccharomyces cerevisiae CEN. . Compared to PK2-1D, the GSH content in the bacterial cells is about 200% to 300% or more, which is very excellent in glutathione-producing ability, and particularly compared to CJ-37, GSH-producing ability is improved by about 584%. was confirmed.

Therefore, since it was confirmed that the CJ-5 strain has excellent GSH-producing ability, it can be seen that the strain of the present application is useful for GSH production.

Confirmation of 18S (ITS, 5.8S) rRNA of CJ-5 strain by gene sequencing Confirmation of the classification of the CJ-5 strain with the highest glutathione production and the CJ-37 strain with the lowest glutathione production isolated in Example 1 To do so, comparative analysis of 18S (ITS, 5.8S) rRNA nucleotide sequences was performed.

Specifically, the 18S (ITS, 5.8S) ribosomal RNA (rRNA) nucleotide sequences of the strains were compared for similarity by BLAST database (Non-Patent Document 12). The 18S (ITS, 5.8S) rRNA sequence of the CJ-5 strain is designated as SEQ ID NO: 1, the 18S (ITS, 5.8S) rRNA sequence of the CJ-37 strain is designated as SEQ ID NO: 2, and the Saccharomyces cerevisiae YJM1592 chromosome XII 18S (ITS , 5.8S) The rRNA sequence was SEQ ID NO:3.

As a result, the CJ-5 strain showed about 93.73% similarity with the Saccharomyces cerevisiae YJM1592 chromosome XII 18S (ITS, 5.8S) rRNA sequence. On the other hand, the CJ-37 strain also showed approximately 95.20% similarity with the Saccharomyces cerevisiae YJM1592 chromosome XII 18S (ITS, 5.8S) rRNA sequence.

Therefore, it was confirmed that both the CJ-5 strain and the CJ-37 strain are Saccharomyces cerevisiae strains. , not only the strain of Saccharomyces cerevisiae CEN. Compared to PK2-1D, the GSH-producing ability is analyzed to be 284%, which is approximately three times higher.

These results show that the strain of the present application has a high GSH-producing ability, and the strain, its dried product, extract, culture and/or crushed product are useful for various cosmetics, foods, feeds, etc. using GSH as a raw material. It suggests that there is

Production of Compositions Containing Glutathione To produce a yeast extract containing glutathione, Saccharomyces cerevisiae CEN. PK2-1D, CJ-5 and CJ-37 yeast cultures were first centrifuged in a centrifuge.

The centrifuged yeast was washed and centrifuged twice to obtain the yeast. Then, a cell wall-degrading enzyme, a nuclease, a nucleotransferase, and a protease were added, and the enzymatic reaction was carried out at pH 5.2 and a temperature of 45° C. for 48 hours.

After the enzymatic reaction described above, the enzymes in the supernatant were inactivated by heat treatment at 85° C. for 30 minutes, and the enzyme-inactivated supernatant was concentrated to prepare a yeast extract.

As a result, Saccharomyces cerevisiae CEN. PK2-1D, CJ-5 and CJ-37 yeast extracts contained glutathione at levels of 0.8%, 2.2%, 0.4% by total weight.

Sensory Evaluation of Food Composition Containing Glutathione The food composition containing the yeast extract produced in Example 4 was subjected to sensory evaluation. Specifically, the Saccharomyces cerevisiae CEN. For the sensory evaluation of PK2-1D, CJ-5 and CJ-37 yeast extracts, consommé chicken soup was used as the basis for sensory evaluation.

A consommé chicken soup mixture containing 5% of each of the above yeast extracts was prepared, and sensory evaluation was performed by sensory inspectors. The evaluation was made in terms of persistence of taste (rich taste), taste (umami), bitterness (abnormal taste), degree of improvement in chicken taste, mellow and deep taste, and overall palatability.

As a result, the CJ-5 yeast extract was found to be CEN. It was confirmed that the PK2-1D yeast extract and the CJ-37 yeast extract were superior in the persistence of taste (rich taste) and mellow and deep taste, and it was confirmed that the overall preference was increased (Fig. 1 ).

Therefore, it was confirmed that the yeast extract of the present application is useful for producing a food composition having excellent taste. and glutathione recovered from at least one of the strain, dried product, extract, culture, and crushed product are also useful for the production of food compositions. .

Confirmation of Antioxidant Effect of Composition Containing Glutathione Example 6-1: Evaluation of Radical Scavenging Ability The radical scavenging ability of the glutathione-containing yeast extract was evaluated.

Specifically, Saccharomyces cerevisiae CEN. Add 5 mL of 0.1 mM 1,1-Diphenyl-2-picrylhydrazyl to 5 mL of yeast extract suspension of PK2-1D, CJ-5 and CJ-37 and react in the dark for 30 minutes, then absorbance at 517 nm was measured. In the concentration range of 2-10 mg/mL, CEN. PK2-1D, CJ-5 and CJ-37 extracts showed radical scavenging effects of 21-61%, 40-83% and 12-49%, respectively (Fig. 2). At a concentration of 10 mg/mL, the antioxidant activity of glutathione-rich CJ-5 yeast extract was reported by CEN. A maximum of 1.7-fold increase in PK2-1D and CJ-37 yeast extracts was confirmed.

Example 6-2: Evaluation of Active Oxygen Absorption Capacity In order to evaluate the antioxidant capacity of the glutathione-containing yeast extract, the Oxygen Radical Absorbance Capacity (ORAC) was analyzed.

Specifically, Saccharomyces cerevisiae CEN. Five milliliters of yeast extract suspensions of PK2-1D, CJ-5 and CJ-37 were mixed with 40 milliliters of Fluorescein and then allowed to react at 37° C. for 15 minutes. 25 mL of 2,2'-azobis-(2-amidinopropane)-HCl was added, after which absorbance was measured at 485 nM and 528 nM for 60 minutes. The active oxygen absorption capacity was compared by determining the area under the absorbance curve (AUC). The active oxygen absorption capacity of CJ-5 yeast extract was reported in CEN. It was higher than PK2-1D yeast extract and CJ-37 yeast extract (Fig. 3). As mentioned above, the antioxidant capacity of the CJ-5 yeast extract of the present invention was evaluated by CEN. It was significantly higher than PK2-1D and CJ-37 yeast extracts.

Therefore, since it was confirmed that the yeast extract has excellent antioxidant ability, the yeast strain, the dried product, extract, culture, crushed product of the strain, and the strain, dried product, extract , culture, and glutathione recovered from at least one of the lysates are useful for antioxidation, detoxification and immune enhancement.

Production Example 1: Production of Food Composition Containing Yeast Extract In the experimental examples described above, it was confirmed that the umami, off-taste, and off-odor control effects peculiar to yeast extracts were confirmed. manufactured.

Specifically, the yeast extract is mixed at a content ratio of 3 to 30% yeast extract, 20 to 40% ammonium chloride, 20 to 40% maltodextrin, 1 to 10% powdered glucose, and 0.1 to 1% emulsifier. A powder was produced.

From the above description, those skilled in the art to which this application belongs will understand that this application can be implemented in other specific forms without changing its technical concept or essential features. . It should be understood that the above examples are illustrative only and not limiting. The present application should be construed to include all modifications and variations that come within the meaning and scope of the claims and their equivalents, rather than the specification.

Claims (15)

A Saccharomyces cerevisiae strain with accession number KCCM12568P that produces glutathione. 2. The strain according to claim 1, wherein the strain comprises the base sequence of SEQ ID NO: 1 as the 18S rRNA sequence. A method for producing glutathione, comprising the step of culturing the strain of claim 1. Claim 3, further comprising recovering glutathione from at least one material selected from said cultured strain, dried product of said strain, extract of said strain, culture of said strain, and homogenate of said strain. Glutathione production method according to. culturing the strain of claim 1;
A method for producing a glutathione-containing composition, comprising mixing at least one substance selected from the cultured strain, its dried product, extract, culture, crushed product, and glutathione recovered therefrom with an additive. 6. The method for producing a glutathione-containing composition according to claim 5, wherein the additive comprises an excipient or an emulsifier. The weight of at least one substance selected from the cultured strain, its dried product, extract, culture, crushed product, and glutathione recovered therefrom is 3 to 30% by weight relative to the total weight of the composition. A method for producing a glutathione-containing composition according to claim 5. The strain according to claim 1, the dried product, extract, culture, crushed product of the strain, and glutathione recovered from at least one of the strain, dried product, extract, culture, crushed product. A composition for antioxidant function comprising at least one substance selected from the group. The strain according to claim 1, the dried product, extract, culture, crushed product of the strain, and glutathione recovered from at least one of the strain, dried product, extract, culture, crushed product. An antidote composition comprising at least one substance selected from the group. The strain according to claim 1, the dried product, extract, culture, crushed product of the strain, and glutathione recovered from at least one of the strain, dried product, extract, culture, crushed product. An immunopotentiating composition comprising at least one substance selected from the group. The strain according to claim 1, the dried product, extract, culture, crushed product of the strain, and glutathione recovered from at least one of the strain, dried product, extract, culture, crushed product. A cosmetic composition comprising at least one substance selected from the group. The strain according to claim 1, the dried product, extract, culture, crushed product of the strain, and glutathione recovered from at least one of the strain, dried product, extract, culture, crushed product. A food composition comprising at least one substance selected from the group. The strain according to claim 1, the dried product, extract, culture, crushed product of the strain, and glutathione recovered from at least one of the strain, dried product, extract, culture, crushed product. A feed composition comprising at least one substance selected from the group. The strain according to claim 1, the dried product, extract, culture, crushed product of the strain, and glutathione recovered from at least one of the strain, dried product, extract, culture, crushed product. A composition for manufacturing a pharmaceutical used for the prevention or treatment of diseases caused by glutathione deficiency, comprising at least one substance selected from the group. The strain according to claim 1, the dried product, extract, culture, crushed product of the strain, and glutathione recovered from at least one of the strain, dried product, extract, culture, crushed product. A pharmaceutical composition for prevention or treatment of diseases caused by glutathione deficiency, comprising at least one substance selected from the group.

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